Bartonella bacilliformis (Bb) is a highly virulent, sand fly-transmitted bacterium that causes Carrin?s disease in humans. This neglected tropical disease is reemerging in endemic regions of the high Andes and expanding into non-endemic areas (i.e., lower altitudes and more diverse habitats) of Ecuador, Colombia and Peru. The at-risk population is ~1.7 million people in a 56,000 square-mile area of S. America, with incidence rates of 12.7 / 100 person years in endemic regions. Bb infections can be life-threatening, with fatality rates of 40-88%, if left untreated, and ~10% fatality rates following antimicrobial intervention. In non-endemic regions, Carrin?s disease manifests as an acute illness with an ~80% reduction in erythrocyte hematocrit (termed Oroya fever; OF), whereas in endemic regions, angiomatous skin lesions (termed verruga peruana; VP) and chronic bacteremia prevail, effectively creating a human reservoir for the pathogen. Our long-term goal is to eradicate Bb through vaccination and/or by disrupting vector-borne transmission to humans. The recent discovery of a geographically overlapping, closely-related but distinct Bartonella species, designated Bartonella ancashensis (Ba), that is less virulent and only causes VP obfuscates our meager understanding of the epidemiology of Carrin's disease. Moreover, the occurrence of Ba complicates the proper diagnosis and treatment of Carrion?s disease, currently based on a patient?s symptoms, blood smears and blood culture. Considering the coincidence of Ba and Bb in the Andes and reported co-infections of humans with the two pathogens, we hypothesize that Ba: a) infects sand flies, b) can be transmitted by infected flies and c) can be co-transmitted with Bb. To address these hypotheses, we propose the flowing aims: In Aim 1, we will create a low-passage Ba strain that constitutively expresses mCherry protein to permanently tag the bacterium. This will be done by site-directed mutagenesis and allelic exchange targeting a redundant locus in the genome. In Aim 2, we will use live-imaging techniques to analyze sand fly competency to harbor and artificially vector Ba. The first goal will analyze colonization and migration of a low-passage, mCherry+ Ba strain in Lutzomyia verrucarum and Lutzomyia longipalpis sand flies using fluorescence microscopy. The tissue(s) involved, duration of infection, and potential for transovarial transmission will be examined. The potential for differential infectivity will also be examined by using three available Ba strains and sand flies. Coinfections of sand flies with mCherry+ Ba and GFP+ Bb will also be assessed. Finally, the ability of Ba Bb to be transmitted / co-transmitted mechanically from artificially-infected human blood in a glass/membrane feeder to uninfected blood in a second feeder will be analyzed. Results of this study will help clarify the epidemiology of these coincidentally occurring pathogens and pave the way for subsequent work investigating their transmission to humans and the molecular basis for their disparate virulence potential.